There is yet no clear explanation of how the separation of chromosomes is achieved by the mitotic apparatus since we lack comprehensive information about how tubulin is assimilated into and disassembled from the kinetochore and non-kinetochore microtubules, how microtubule assembly is locally and selectively controlled within the spindle, how the forces for chromosome movement are generated, and how the assembly and disassembly of the microtubules govern the production and regulation of chromosome movements. We have few answers to these fundamental questions because of our inability to isolate in a cell-free preparation a mitotic apparatus that retains the capacity for microtubule assembly and chromosome movements exhibited in vivo. My preliminary studies of sea urchin spindles located in low ionic strength buffers containing EGTA and Triton X-100 show promise and need to be extended and expanded. These studies also have pointed out the possible role of Ca ions and Ca ions-sequestering systems in the cellular control of spindle microtubule assembly and functions. This possibility deserves further examination not only on spindle isolates but in mitotic spindles in vivo. Additionally, the isolated spindles contain significant amounts of actin which provides us the opportunity to explore the physiological relevance of actin-myosin contractility to chromosome poleward movements. Newly developed procedures for labelling tubulin with fluorescent substances will be used to track the flow of tubulin molecules in, out, and through the spindle microtubules to understand better the intimate coupling of microtubule assembly and disassembly to the movements of chromosomes. I also propose to explore further the effects of sudden changes in hydrostatic pressure and temperature on kinetochore microtubule depolymerization and anaphase chromosome movements in Crane Fly spermatocyte cells. Preliminary evidence has indicated that kinetochore microtubules are not simply in a reversible equilibrium, but are in a dynamic equilibrium resulting from a steady-state balance of enzymatically-controlled irreversible microtubule assembly and disassembly reactions.